Understanding the association of iris colour and myopia may provide further insights into the role of the wavelength of lights in the pathophysiology of myopia. We aim to assess the association of iris colour and myopia in a school-based sample of Chinese students. Two thousand three hundred and forty-six Year 7 students from 10 middle schools (93.5% response rate) aged 13-14 years in Mojiang, a small county located in Southwestern China, participated in the study. We obtained standardised slit lamp photographs and developed a grading system assessing iris colour (higher grade denoting a darker iris). Refractive error was measured after cycloplegia using an autorefractor by optometrists or trained technicians. An IOLMaster (www.zeiss.com) was used to measure ocular biometric parameters including axial length (AL). Of all the study participants, 693 (29.5%) were affected by myopia with the prevalence estimates being higher in girls (36.8%; 95% confidence interval [CI]: 34.0, 39.6) than in boys (22.8%; 95% CI: 20.4, 25.1) (p < 0.001). After adjusting for gender, height, parental history of myopia, time spent on computer, time spent watching TV, time spent outdoors, and time spent reading and writing, participants with a darker iris colour tended to have a higher prevalence of myopia, a more myopic refraction and a longer AL. Dose-response relationships were observed in all regression models (p for trend <0.05). Darker iris colour was associated with more myopic refractive errors and longer ALs among Chinese school-aged children and this association was independent of other known myopia-related risk factors.